Hydrophilic interaction chromatography (“HILIC”) separation coupled with fluorescence detection in UPLC platforms can be used to resolve complex N-linked glycan populations due to the ability to separate both neutral and charged glycans in a single chromatographic run. Because of the heterogeneity, isomerization and anomericity of glycans, elucidating the structure of complex N-linked glycans can be a significant analytical challenge. A dextran ladder can be used to calibrate hydrophilic interaction chromatography (“HILIC”) separations of glycans and convert peak retention times into glucose unit (“GU”) values. Each individual glycan structure has a GU value which is directly related to its linkages and its constituent monosaccharides. The GU value can be used to predict structures because each glycan structural component contributes in a specific way to the GU value of a given glycan.
As such, the elution times of glycans can be expressed in glucose units by reference to a separation calibrant, such as one based on a dextran ladder. A dextran ladder can be used to calibrate the liquid chromatography (“LC”) runs against day-to-day or system-to-system changes. A GU value can be calculated by fitting a fifth order polynomial distribution curve or a cubic spline fit to the retention times of the dextran ladder. Then, using this curve, GU values can be assigned from the retention times observed in a test sample. The GU values for N-glycans can be reproducible, with inter-column standard deviations less than 0.3. This allows direct comparison with database values collected from a range of instruments over a period of time. Having GU values, databases of glycans stored in values of GU can be interrogated to aid in elucidating the potential glycan structures existing with a glycan population.
Furthermore, even when comparing different approaches for tagging, it can be seen that the labeled N-glycans are resolved by the HILIC separation into very similar profiles. It is not a trivial task to produce a dextran ladder that is appropriate for use with glycans that are rapidly labeled with a rapid tagging reagent. Dextran is a saccharide comprised of a reducing, aldehyde terminus, different than N-glycosylamines which are released via enzymatic deglycosylation of N-glycosylated proteins. Unlike the latter, dextran cannot therefore be modified with a rapid tagging reagent that targets amine nucleophiles. The dextran cannot be rapidly labeled because the saccharide does not contain an appropriate nucleophile. A need exists, as a result, for methods of calibrating the liquid chromatography processes with a standard that has the same optical and/or other physiochemical properties as the rapid labeled N-glycan.